No support for socio-physiological suppression effect on metabolism of paired white mice (Mus sp.)

Authors


Abstract

1. Three experiments were performed on white mice (strain MF1) to establish if there was social or physiological suppression of metabolism, mediated by elevated ambient levels of CO2, when animals were in close contact. In the first two experiments the metabolism of two mice were each measured solitarily and paired together in the same chamber, both with and without a partition that allowed visual, auditory and olfactory communication but restricted physical contact.

2. Comparisons were made between the sum of the two solitary measurements, and the observed measurements of paired individuals. In the first experiment air flow was low and ambient CO2 was always at levels predicted to produce suppression (> 0·2%), independent of whether the animals were measured alone or paired.

3. The paired mice had greater metabolism than expected from the sum of their solitary metabolic rates whether the partition was present or absent. This increase was due to increased activity of the animals when in pairs. This experiment indicated that when CO2 levels were maintained in the range 0·2–0·5% there was no social suppression of metabolism.

4. In experiment 2 metabolic rates were measured of solitary mice exposed to subthreshold levels of CO2 (≈ 0·12%) and paired mice that experienced levels above the supposed threshold (≈ 0·24%). Again the observed metabolism of the paired mice exceeded the sum of their metabolic rates when solitary. The increase was also attributable to greater activity of the animals in pairs.

5. This experiment indicated no social suppression effect when CO2 varied in the range 0·12–0·24%. Moreover there was no significant difference between the oxygen consumptions of mice in experiment 2 compared with experiment 1, indicating that independent of the social context there was no suppressive effect of increases in CO2 concentration over the range 0·12–0·5% CO2.

6. Finally, in experiment 3, when two mice were placed in sequential separate chambers (allowing no social contact), their summed metabolism did not differ from the sum of their solitary measurements when a CO2 absorber was placed in the airflow linking the chambers. When the CO2 absorber was absent the observed metabolism of the pair exceeded the sum of the solitary measurements.

7. Overall, across all three experiments no evidence was found for either a social or a physiological suppression of metabolism mediated via CO2 (0·12–0·5%).

Introduction

Small mammals often huddle together in groups. The reasons for huddling behaviour may be diverse. However, theoretical models based on the geometry of exposed surface areas (e.g. Canals, Rosenmann & Bozinovic 1989, 1997), and direct measurements of the energy expended by animals grouped together in huddles (Andrews & Belknap 1986; Bryant & Hails 1975), suggest that an inevitable consequence of huddling may be that the energy expenditures of the individual animals in the huddle are reduced when compared with solitary individuals. This energy saving may itself be a selective advantage favouring the evolution of huddling behaviour in certain circumstances (Sealander 1952; Fedyk 1971; Gebczynska & Gebczynski 1971; Baudinette 1972; Karasov 1983; Springer, Gregory & Barrett 1981).

The physical basis for the reduction in energy expenditure of huddled animals has been the focus of some debate. On one hand the effect may be due to reductions in the mean individual exposed surface area of huddled vs solitary animals (Contreras 1984; Vickery & Millar 1984; Canals et al. 1989). Alternatively huddles of animals may elevate the local temperatures more effectively than solitary animals, meaning the local temperature experienced by the huddle is greater than that experienced by solitary individuals (Andrews, Phillips & Makihara 1987; Hayes, Speakman & Racey 1992a). Hayes et al. (1992a) partitioned these effects, controlling for differences in activity between huddled and solitary Short-Tailed Field Voles (Microtus agrestis), and found that approximately half the observed saving could be attributed to each effect. Canals et al. (1997) suggested the majority of the effect was accounted for by reduced surface area.

Apart from the physical effects of huddling on energy expenditure there may also be direct biological effects of animals upon each other when they are in close proximity. Thus two animals huddling together may have a calming effect on each other and mutually reduce each other’s metabolic rates. For example, Martin, Fiorentini & Connors (1980) found that single white mice (Mus sp.) and gerbils (Meriones unguiculatus) had greater metabolic rates than huddled trios in the same chamber, or of the same three animals separated by partitions to prevent huddling. In direct contrast to these findings, however, Contreras (1984) found no suppressive effect on metabolism when trios of mice and gerbils were measured together but separated by partitions, indicating no social effect on metabolic rate.

In part the different results found by Martin et al. (1980) and Contreras (1984) may reflect subtle differences in experimental design and the fact that in some circumstances there may be more direct physiological effects of one animal on the metabolism of its immediate neighbours. Herreid & Schlenker (1980) found that if a mouse was placed in a completely separate chamber upstream of a second mouse, the metabolism of the second mouse was suppressed for the duration that the two chambers were connected. These experiments led them to suggest that an airborne factor was responsible for suppressing the metabolism of the second mouse.

In a second paper, Schlenker & Herreid (1981) performed some further manipulations using the same system to try to establish the nature of the airborne factor. The conclusion of this work was that the suppressing factor was elevated levels (0·2–0·8%) of CO2 in the second chamber owing to exhaled CO2 from the first mouse. In further experiments, Schlenker, Carlson & Herreid (1981) demonstrated that the effect was still present in anosmic mice, indicating that the effect was mediated by CO2 and not some other olfactory cue.

Observations of socio-physiological effects of small mammals on each other have several potential implications for observations of energy savings when animals come together in huddles. In particular, at least part of the observed saving may be due to social and physiological effects rather than any physical effect due to reduced surface area or local heating. Whether animal metabolic rates are reduced simply by the presence of conspecifics, or whether suppression is additionally mediated physiologically by low ambient levels of CO2 is an important question that has ramifications not only for our understanding of why energy savings might occur in huddled animals, but also for respirometry measurements of energy expenditure in general. Measurements of resting and basal metabolic rates, for instance, are always made for animals that are solitary, and, if an open flow system is employed, also in the CO2-rich atmosphere of a respirometry chamber. If there is a ‘calming’ effect when other individuals are present that is absent if the animal is alone, this may have ramifications for the interpretation of the practical significance of BMR and RMR measurements. In particular, are solitary animals in the wild in the ‘calm’ state, or in the same state as that pertaining in measurement protocols for BMR and RMR? Alternatively, does the artificially high level of CO2 in a respirometry chamber lead to suppressed metabolism relative to that normally experienced by animals in the wild? These potential effects may contribute to the reported discrepancies between time and energy budget calculations of daily energy expenditures of animals, and more direct measurements based on the doubly labelled water technique (Weathers et al. 1984; Nagy 1989). In the current paper we report a series of experiments in which we aimed to assess the effects of social and physiological suppression on the metabolism of white mice.

Methods

GENERAL DETAILS

We performed experiments on white mice (Strain MF1) housed separately in plastic cages (30cm× 15cm×12cm) at 20°C, 12L:12D, with water and food (Special Diet Services CRM; BP Nutrition Ltd, Wytham, Essex, UK) available ad libitum. All the experiments involved standard respirometry measurements of oxygen consumption using a system that has been previously described (Speakman & Racey 1988; Hayes et al. 1992a,b). In experiments 1 and 2 a single Perspex respirometry chamber measuring 25 × 25 × 12 cm was used, with sawdust in the base to absorb urine. The chamber could be divided into two sections by inserting a Perspex sheet down the centre. The divide had 16 5-mm holes drilled into it. There was consequently a free passage of air across it, and also, as it was Perspex, the animals could see each other through it although they could not make physical contact. A temperature probe was inserted near the air outlet. While in the chamber the mice were not allowed to eat or drink but were not food deprived prior to entry. All the measurements were made at a thermoneutral temperature of 30 °C (lower critical temperature in these mice is 28 °C). Measurements wre made at thermoneutrality to eliminate any physical thermal effects of huddling in the paired mice. Any observed suppression of metabolism could thus be attributed to social or physiological factors. In experiment 3 two separate chambers linked in series were used. Barometric pressure readings were taken at the start of measurements and the temperature inside the flowmeter monitored throughout the run. All oxygen consumption estimates were corrected to STP.

In all three experiments observations were made on single sex groups of five individuals. It had originally been planned to examine the role of factors such as gender on the extent of any socio-physiological suppression. However, it was found that whenever two mice of opposite sex were placed together they almost always immediately fought or copulated – both of which compromised the estimation of a resting metabolic rate. In addition once females became pregnant they changed their metabolism over time, thus also compromising the experimental design which involved sequential pairings of individuals over a period of days. After these observations the design was restricted to single sex groups of females. The animals in each group of five were exhaustively paired together with all other members of their group (n = 10 pairings). In experiments 1 and 2 for each pairing measurements were made of metabolic rate (oxygen consumption) in four different conditions: (1) Mouse A alone; (2) Mouse B alone; (3) Mice A and B both in the chamber together with no divide; and finally (4) Mice A and B both in the chamber but separated by the divide. As patterns of circadian and ultradian rhythms in metabolism have been observed in a number of small laboratory animals (Lehmann 1976; Kleinknecht, Erkert & Nelson 1985; Stupfel et al. 1987) including mice (Mount & Willmott 1967) the order of the conditions was randomized. In conditions (1) and (2) the partition was present so that the animal was allowed to stay only in one half of the respirometry chamber; the side of the chamber used in each case was randomly decided. In condition (4) allocation of the animals to each side of the divide was randomized. Between each condition the chamber was cleaned thoroughly and the sawdust changed.

Measurements were made in each condition for 90 min. The first 30 min was considered a settling period. Behavioural recordings were made in the last 60 min, after which the animals were taken out of the chamber and the analysers allowed to stabilize at ambient levels for 30 min before the start of the next measurement. During the 60 min of behaviour recording the mice were observed continuously and their behaviour logged at the end of each minute. Behaviour was divided into three classes: resting, grooming and general activity. When two mice were placed together in a chamber, without the partition, allogrooming would occur; this was included in the grooming category. General activity included walking, digging and chewing the sawdust, investigating the chamber and, when the mice were placed together, any social interactions that might occur apart from allogrooming. The locations of each individual, and thus their distances apart, were also recorded each minute. When the animals were together in the unpartitioned chamber, one individual was marked by touching it briefly on the back with a water-soluble marker pen, prior to the start of the measurements. This had no apparent effect on behaviour (no difference between marked and unmarked individuals in their time budgets).

EXPERIMENT 1: LOW FLOW RATE

In this experiment 10 mice were used. They were randomly divided into two groups of five and exhaustively paired within their own group. Each pairing was used for 1 day’s measurements. The air flow used in this experiment averaged 602 ml min–1. The flow was not varied within each condition, and thus the ambient CO2 levels were greater during the period when two mice were in the chamber compared with only single mice. CO2 content was not monitored directly throughout these experiments as a CO2 analyser was not available at the time the measurements were made. In a separate group of the same strain of mice feeding on the same diet the respiratory quotient (RQ = CO2 production divided by O2 consumption) was measured using in-line CO2 and oxygen analysers. Steady-state RQ averaged 0·953 (SD = 0·04, range 0·91–0·99, n = 7). Thus for this strain of mice feeding on this diet the CO2 concentrations in the chambers would be ≈ 95% of the observed percentage reduction in oxygen concentration.

Given our determinations of RQ and the flow rate used in this experiment the CO2 content of the chamber was on average 0·22% when single mice were present and 0·44% when two mice were present. Thus CO2 content was always higher than 0·2%, and always in the range shown previously by Schlenker & Herreid (1981) to produce suppression. Since at thermoneutrality the possibility of any physical benefits of sociality can be discounted, any suppression of metabolism in the paired conditions could thus be attributed to a social ‘calming’ effect, and any difference between conditions (3) and (4) might point to how this effect was mediated.

Two quantifications of metabolic rate were made. First, the mean oxygen consumption was measured over the same 60 min for which behavioural observations were made. Second, the RMR was measured as the lowest consecutive 5 min of oxygen consumption throughout the entire run.

EXPERIMENT 2: HIGH FLOW RATE

The protocol for the second experiment was identical to that of the first, but a new group of 10 mice was used, and the average flow of air though the system was increased to 1113 ml min–1. The increase in air flow between this experiment and the previous one was estimated to be enough to eliminate any possible self-inhibition of metabolic rate for the solitary mice. In the solitary conditions the ambient CO2 level was estimated to be 0·12% but in the paired conditions it was estimated to average 0·24%. These levels therefore crossed the boundary at which Schlenker & Herreid (1981) suggested that suppression becomes evident (0·2%).

It was anticipated that if suppression due to CO2 occurs this would be detectable in two ways. First there would be a reduction in the metabolism of the paired individuals relative to the sum of their solitary metabolic rates, the extent of which would be independent of the presence or absence of the partition. Second, the metabolic rates of the mice in this experiment would be expected to be higher than the metabolic rates of mice in the first experiment under identical social conditions.

EXPERIMENT 3: SEPARATE CHAMBERS LINKED IN SERIES

In the third experiment two separate cylindrical chambers (A and B) were used, each consisting of a Perspex tube (30 cm long, 7 cm in diameter) closed at either end by a rubber bung. In each chamber a perforated plastic floor (6·5 cm × 25 cm) was fitted to keep the animals separate from their faeces and urine. Air inlet and outlet were at opposite ends of the chamber and a temperature probe was inserted near the outlet. The chambers were connected in series and the tube connecting them could be substituted with a Perspex tube filled with sodium hydroxide pellets and silica gel to remove the CO2 produced by the mouse in the first chamber and prevent it from reaching the mouse in the second chamber.

In this experiment a further 10 mice were used, and they were divided into two groups as in experiments 1 and 2, but the conditions they were exposed to were different:

1. Mouse in chamber A (upstream), with CO2 absorber between chambers.

2. Mouse in chamber B (downstream), with CO2 absorber between chambers.

3. Mice in chambers A and B, with CO2 absorber between chambers.

4. Mice in chambers A and B, without CO2 absorber.

The rationale for this protocol was to investigate the physiological effect of CO2 alone, taking out any effects of social factors by keeping the two mice completely isolated with respect to social contact. Each mouse was randomly assigned to either chamber A or B, and was placed only in that chamber for that day’s recordings. As in previous experiments, the order of conditions was randomized. The same definitions were used for mean oxygen consumption and RMR as employed in the previous two experiments.

STATISTICS

In all three experiments a comparison was made between the summed oxygen consumption of two mice measured solitarily (generally termed the predicted summed oxygen consumption) and that measured when they were together. The animals were assigned to groups of five individuals which were exhaustively paired together (giving a sample size of 10 comparisons). Since in all three experiments there were two groups of five individuals, there was a total of 20 comparisons between the summed prediction and the observed oxygen consumption of paired individuals for each experiment. The means were compared across these 20 comparisons using paired t-tests. The null hypothesis in each case was that there would be no difference between the summed measurements and the measurements of the animals together. Although a priori we were interested in suppression, two-tailed tests were made because our null hypothesis was that there was no effect, and we did not want to ignore the possibility that paired mice might mutually stimulate each other’s metabolic rates. As several repeated tests were made within each experiment, the significance levels were adjusted for the number of comparisons being made using the Bonferoni correction. Cited significance levels (0.05 and 0.01) include the correction for repeated testing.

A more refined prediction was also generated which controlled for changes in the activity of the animals between the separated and paired conditions. To do this the measurements of oxygen consumption and simultaneous records of the behaviour of solitary individuals were used. Body mass and percentage of the time spent resting were entered as predictors of the oxygen consumption in a regression analysis. The expenditure of each individual in the paired condition was then predicted by substituting the known mass and percentage time spent resting for that individual into the equation, and summing across the two individuals. The derived prediction was tested in the same way as the simpler prediction which did not take into account changes in the behaviour, i.e. using a paired t-test. The predictions were made both with, and without, arcsine square root transformations of the percentages. This transformation made no difference to the significance of the effects.

In addition to paired comparisons within each experiment the oxygen consumptions of mice between experiments 1 and 2 were also compared. As different mice had been employed in these experiments metabolic rates in each condition were compared by two-sample t-tests. For the solitary comparisons single observations of each individual from each group were chosen to avoid pseudo-replication. Again the null hypothesis was that there was no difference and two-tailed tests were employed.

The significance of behavioural changes between conditions was explored using a χ2-test. The behaviour of the two individuals when solitary was combined to derive an expectation, and this was compared with the actual observed behaviours when the animals were together. All tests were performed using the MINITAB statistics package (Ryan, Joiner & Ryan 1985).

Results

EXPERIMENTS 1 AND 2: LOW AND HIGH FLOW RATES

The average mass of the mice in experiments 1 and 2 was 33·3 g (SD = 2·7 g) and 33·6 g (SD = 4·7 g), respectively. In both experiments the observed mean oxygen consumption (average over last 60 min of measurements) for paired individuals was significantly higher than the predicted summed oxygen consumption of the solitary animals, both with and without a partition (Fig. 1). The presence or absence of the partition had no significant effect on the metabolism of the paired animals (P > 0·05 in both experiments).

Figure 1.

. Histogram comparing mean oxygen consumptions (average over last 60 min of measurements) of white mice measured solitarily (one and two indicate first and second mouse measured, respectively), the sum of these individual measurements (predicted) and measurements of paired mice with a partition (+ partition) and paired without a partition (no partition). Results (a) from experiment 1 with a low flow rate through the chamber and (b) from experiment 2 with a high flow rate through the chamber. Error bars reflect SD (n = 20 in all cases).

The RMR (lowest over 5 min) of solitary individuals, predicted combined measurements, and the observed paired conditions, followed the same trend as the mean oxygen consumptions (average over last 60 min). The predicted summed RMR was significantly lower than the observed RMRs of paired individuals both with or without a partition (Fig. 2). The RMR of paired individuals without the partition did not differ significantly from the measurements with the partition (P > 0·05 in both experiments).

Figure 2.

. Histogram comparing resting metabolic rates (minimum reported oxygen consumption over 5 consecutive minutes throughout entire run) of white mice measured solitarily (one and two indicating the first and second mouse measured, respectively), the sum of these individual measurements (predicted), and measurements of paired mice with a partition (+ partition) and paired without a partition (no partition). Results (a) from experiment 1 with a low flow rate through the chamber and (b) from experiment 2 with a high flow rate through the chamber. Error bars reflect SD (n = 20 in all cases).

In these experiments the solitary individuals were significantly less active, i.e. they spent a greater proportion of their time resting, than the paired individuals (Table 1). In both experiments grooming was performed less frequently than general activity. These behavioural differences between measurement conditions were highly significant (χ2-values 731·5 and 370·1 for experiments 1 and 2, respectively). The distance, to the nearest cm, between the two mice in the condition without a partition was 6 cm for both experiments. When allowed physical contact the mice spent on average 55% (SD = 27%) and 70% (SD = 27%) the time huddling together in experiments 1 and 2, respectively.

Table 1.  . Behavioural observations of mice in experiments 1 to 3. In experiments 1 and 2 the mice were in a single chamber either alone or paired, with or without a partition. The air flow rate was about double in experiment 2 relative to that in experiment 1, thus ambient CO2 levels were about halved in the experiment 2. In experiment 3 the mice were in two separate chambers linked in series. In all cases f refers to the frequency which is the summed frequencies of observations of that behaviour across all 20 replicates in that condition (n = 60 observations per replicate).% refers to the percentage representation of that behaviour in that experiment. For experiments 1 and 2, First refers to the first mouse into the respirometer on its own, and Second refers to the second mouse into the respirometer on its own. + Part refers to measurements made on mice with a partition separating them, and – Part refers to measurements made on mice free to associate. The sample sizes in these latter two conditions are double because two mice contribute to the observations. For experiment 3 the notation A refers to a single individual in the upstream chamber with no mouse downstream, B refers to a single individual in the downstream chamber with no mouse upstream, AB + refers to two mice in the chambers with a CO2 absorber separating them, and AB – refers to two mice in the chambers without the CO2 absorber Thumbnail image of

In both these experiments there was a significant negative relationship between the mean oxygen consumption for single individuals, measured over the last 60 min of the measurement period, and percentage of the same interval spent resting (Fig. 3), the correlation coefficients were – 0·79 and – 0·83, respectively. Regression analyses were performed using oxygen consumption as the dependent variable and percentage time resting and body mass as independent predictors. In experiment 1 67·5% of the variation in oxygen consumption of single mice could be accounted for these factors. In experiment 2 the same two independent predictors explained 74·8% of the variation in mean oxygen consumption of single mice.

Figure 3.

. Mean oxygen consumptions over the last 60 min of measurement of solitary mice plotted against the percentage of the same interval spent at rest. Results for experiments (a) 1 and (b) 2 are shown. In both cases there was a significant negative relationship (fitted lines)

The regression equations were used to predict the mean oxygen consumption for the paired individuals. These predictions take into account any differences in the activities of the animals between the paired and unpaired conditions. When these differences were accounted for, the difference between the observed oxygen consumption of paired individuals and that predicted from summing the two solitary measurements disappeared (paired t-tests: experiment 1 t=–0·89, P > 0·05; experiment 2 t = 1·72, P > 0·05). This indicates that the elevated oxygen consumption of paired mice, relative to the sum of their solitary measurements in both these experiments was a result of increased activity in the paired conditions (Table 1).

The resting oxygen consumptions observed in experiment 1 were compared with those observed under identical social conditions in experiment 2. For all four conditions there was no significant difference between the two experiments (mouse 1 alone t = 0·85, P > 0·05; mouse 2 alone t = 0·34, P > 0·05; mice 1 and 2 together with a partition, t = 0·94, P > 0·05; and mice 1 and 2 together without a partition t = 0·13, P > 0·05).

EXPERIMENT 3: SEPARATE CHAMBERS IN SERIES

In the third experiment, where the mice were kept in separate chambers, the predicted summed mean oxygen consumption was not significantly different from measurements of paired individuals with a CO2 absorber between them (paired t-test = 0·02, P < 0·05). However, the oxygen consumption of paired mice without a CO2 absorber was significantly greater than predicted from the summed solitary measurements (paired t-test = 2·78, P < 0·01) (Fig. 4) and the paired measurements with an absorber (t = 2·63, P < 0·01).

Figure 4.

. Histogram comparing mean oxygen consumptions (average over last 60 min of measurements) of white mice measured solitarily (one is in the upstream chamber and two is in downstream chamber), the sum of these solitary measurements (predicted) and measurements of the mice simultaneously in the two chambers in series separated by a CO2 absorber (+ Absorber), and with the CO2 absorber removed (No absorber). Error bars reflect SD (n = 20 in all cases).

The RMR values for the third experiment had a different pattern from that of the mean oxygen consumption. The predicted sum of solitary measurements of RMR was significantly lower than observed in both the paired conditions, i.e. with and without CO2 absorber (Fig. 5) (paired t-tests: with absorber t = 3·12, P < 0·01; without absorber t = 3·77, P < 0·01). The observed RMR in the paired condition without the CO2 absorber was not significantly different from that with the CO2 absorber (t = 1·04, P > 0·05).

Figure 5.

. Histogram comparing resting metabolic rates (minimum reported oxygen consumption over 5 consecutive minutes throughout entire run) of white mice measured solitarily (one is in upstream chamber and two is in downstream chamber), the sum of these solitary measurements (predicted) and measurements of the mice simultaneously in the two chambers in series separated by a CO2 absorber (+ Absorber), and with the CO2 absorber removed (No absorber). Error bars reflect SD (n = 20 in all cases).

In experiment 3 there was a very low level of activity in all conditions and similar amounts of grooming and general activity within each condition (Table 1). Note that in this experiment the animals were kept in separate respirometry chambers throughout the conditions and there was no physical, visual or acoustic contact between the mice when tested in series. The differences in behaviours between conditions were, however, still significant (χ2 = 156·5, P < 0·01). The paired individuals without the CO2 absorber displayed the highest amount of time spent resting (Table 1).

Since the animals in experiment 3 were less active than the mice in experiments 1 and 2, the relationship between mean oxygen consumption and percentage time spent resting was less significant (r = – 0·659) (Fig. 6), and the regression relating the oxygen consumption to both body mass and percentage time resting only explained 51·9% of the variation in mean oxygen consumption. This equation was used to predict the oxygen consumptions of the mice from behaviour when there was no CO2 absorber between the chambers. The observed oxygen consumption for paired individuals without the absorber was still significantly higher than the predicted oxygen consumption (paired t-test, t = 3·26, P = 0·0041) even after taking activity changes into account.

Figure 6.

. Mean oxygen consumption over the last 60 min of measurement of solitary mice plotted against the percentage of the same interval spent at rest for animals in experiment 3. There was a significant negative relationship (fitted line).

Discussion

EXPERIMENT 1: LOW FLOW RATE

Mice in pairs had greater oxygen consumption than anticipated from the sum of measurements made when they were solitary. This effect occurred independently of whether a partition was present in the chamber or not. The increase was accounted for by the changes in time budgets of the animals involved. Paired individuals were more active than solitary individuals.

Overall these data indicate that when CO2 levels were always in the range expected to produce suppression (cf. Schlenker & Herreid 1981) there was no ‘calming’ social effect on metabolic rate. If anything, the effect was opposite. This result conflicts with the suggested social effect on metabolism by Martin et al. (1980). In that study, however, no behavioural observations were made, and CO2 levels were not regulated to be always within the range found by Schlenker & Herreid (1981) to produce suppression. Consequently the reported ‘social’ effect on the metabolic rates of gerbils and mice, by Martin et al. (1980), might reflect a combination of several effects, such as altered time budgets or physiological suppression due to elevated CO2 concentrations in the paired situations, not necessarily including a ‘social’ suppressive effect. Our data clearly show that no social suppression of metabolic rate occurs when CO2 levels are maintained in the range 0·22–0·44%. Moreover the extent of social contact, i.e. including or excluding physical contact, had no effect on this result. Our data support the observations of Contreras (1984) who found no differences between the metabolism of trios of mice and gerbils when measured together but separated by partitions, or measured solitarily. Contreras (1984) did not attempt to control the CO2 levels experienced by the animals, but recalculation of the probable conditions, from the quoted metabolism and flow rates, suggests that CO2 concentrations in the respirometry chambers were also always above 0·2%.

EXPERIMENT 2: HIGH FLOW RATE

By manipulating the flow rate to high levels in this experiment solitary mice were exposed to levels of CO2 below the suppression threshold suggested by Schlenker & Herreid (1981) and paired mice to greater (potentially suppressive) levels. Despite this no significant suppression of metabolic rates was found in the paired condition. Indeed, the observed metabolic rates in the paired conditions were higher than the prediction, as had occurred in experiment 1, and the effect was similarly attributable to the increased activity of the mice housed in pairs. These data indicate no support for a suppressive effect across this threshold in this strain of mice, and also indicate no social effect occurs at these lower levels of CO2 concentration.

By comparing the metabolic rates of animals in identical social situations between experiments 1 and 2 no effect of ambient CO2 content on the metabolism between 0·12 and 0·44% was also confirmed. These results could occur because no such threshold exists in these animals or perhaps the threshold was at much higher CO2 levels. There is some evidence supporting interspecies differences in the positioning of a threshold for suppression, since Kay (1977) indicated that ambient CO2 levels greater than 1% were necessary to influence the metabolic rates of Banner-Tailed Kangaroo Rats (Dipodomys spectabilis). If this was the case and high levels of CO2 would result in suppression of metabolism in this strain of mice, the threshold would need to be at least 0·5%.

EXPERIMENT 3: SEPARATE CHAMBERS

By placing mice in separate chambers, in series, we hoped to eliminate any social cues (physical, visual or auditory contact) that might facilitate social suppression of the metabolism. In this situation no evidence was found that there was suppression of the metabolism of the second mouse when it was placed in series with the first mouse. The presence or absence of a CO2 absorber had no detectable effect. Indeed when the CO2 absorber was not present the summed metabolic rate of the two individuals appeared to be elevated rather than suppressed.

These data are contradictory to those collected by Herreid & Schlenker (1980) and Schlenker & Herreid (1981) who discovered very clear suppression, of up to 35%, in the metabolic rate of the second mouse when connected with the first mouse upstream using an almost identical protocol. The protocol used by Herreid & Schlenker (1980) was superior to that used by us since they employed two gas analysers to estimate the metabolism of both mice independently, whereas we measured only the summed metabolism of the two individuals.

Given these differences, an unlikely but possible scenario in the present experiments was that the metabolic rate of the second mouse was suppressed but that this was more than offset by an increased metabolism of the first mouse in the condition when both chambers were connected. This seems unlikely because the mass and activity were the dominant factors influencing the metabolism of the individual animals, and the upstream mouse did not change its activity significantly between conditions when there was, and was not, a mouse in the second chamber. Moreover, when the equation taking into account the masses and any changes in activity was employed, the mice in the paired condition had greater oxygen consumption than the predictions. The only mechanism therefore would be for the first mouse to have elevated its resting metabolism, independent of activity, in response to the presence of the second mouse. How the first mouse might detect the second mouse to effect such a change is uncertain. A more plausible scenario was that there was no suppression owing to low levels of CO2 in these animals. This is also consistent with the results of experiments 1 and 2.

A potential problem with all our experiments was that the CO2 levels were not measured directly in the chambers directly but were inferred from a combination of the decline in oxygen percentage levels and the RQ observed in a separate group of animals. In a different series of RQ measurements made 10 years previously, using the same strain of mice but a different commercial diet, lower RQs of around 0·8 were observed (J. R. Speakman, unpublished data). If the RQ in the present experiments had been only 0·8 this would change the interpretation of individual experiments. In experiment 1, solitary mice would have been below the supposed threshold eliciting suppression (0·2%) but paired mice would have been above the threshold. In experiment 2 the mice would have always been below the threshold. In both cases the absence of any effect of pairing on metabolic rates still suggests that neither social factors nor low levels of ambient CO2 are suppressive factors. Our interpretations are therefore robust to the inferred RQ.

The most likely explanation for the discrepancy between the current results and those of Herreid & Schlenker (1980) and Schlenker & Herreid (1981) is that the effect detected by them was specific to the strain of mice they studied (RR mice). Unfortunately, we could not obtain this strain of mice to test this possibility. Another difference between our experiments and some of their protocols was that our mice were always housed alone but in some of their experiments the mice were housed together. The extent to which prior sociality affects the extent of suppression and how widespread the effect they detected is remains open to further investigation.

Conclusions

Across all these experiments, in which the social conditions and the CO2 environment were manipulated, no evidence was found for any significant suppression of the metabolic rates of individuals in pairs, compared with when they were solitary. Where differences in oxygen consumption did occur they were in the direction opposite to that expected from suppression, and were linked to differences in time budgets of the animals between the different conditions. Removing the effects of the differences in time budgets generally removed these effects. We find no support for the notions of social or physiological suppression effects on the metabolic rates of these animals.

Acknowledgements

We are grateful to Prof C.F. Herreid for searching for and providing details of suppliers for the RR mice. This work was supported by NERC grant GR3/5155.

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